The invention relates to a process for the industrial production of ergoline derivatives including alkaloids and 9,10-dihydroalkaloids of the ergotoxine group and including also ergometrine.
These alkaloids and 9,10-dihydroalkaloids are known for their high therapeutic usefulness and are employed in internal medicine, neurology and gynecology.
The industrial recovery of ergot alkaloids up to now has been carried out on a large scale by agricultural growing of alkaloid type sclerotia of the microorganism Claviceps purpurea which exists as a parasite on rye. To avoid the difficulties involved in this type of production attempts have been made to cause the organism to form alkaloids also under non-parasitic conditions. It has been found however that only very few strains of Claviceps were useful for this purpose and only when grown particularly therefor. Only with these few strains and only in specially set-up media could the biosynthesis of ergot alkaloids be effected.
Since the composition of the alkaloid spectrum of these strains depends considerably on the different genetic conditions only a portion of the strains forms alkaloids of the ergotoxine groups which are sufficiently differentiated therapeutically in the general group of ergot alkaloids of the peptide type.
The existence of a few such strains has been described in detail in Hungarian Pat. No. 150,631, patent of the German Democratic Republic No. 41,967, British Pat. No. 1,158,380, and patents of the Federal Republic of Germany, Nos. 1,806,984 and 1,909,216. Nevertheless, the problem of a saprophytic production of alkaloids of the ergotoxine group alone or in combination with other ergot alkaloids has not been solved insofar as production on an industrial scale is concerned.
For a continuous production the requirement is that a high performance starting material of the strain must be currently available in large amounts as inoculum for the fermentation process and that production must be possible in a simple and definite manner.
The customary and best method in technical microbiology is the use of special reproduction units of the organism (conidiospores) which provide for a stable source of the desired properties of the strain throughout many years by means of lyophilized spores containing cans, that is, without the complicated and laborious strain preservation as described in German Pat. No. 1,206,384, but also because the large numbers of conidiospores per volume unit-permits to inoculate large amounts of nutrient medium with a small amount of inoculum.
All this requires a specific characteristic of the strain which is determined by its heritable disposition to form under proper conditions a large number of spores having a high potency for the biosynthesis of the alkaloids. The inoculation of the culture medium can then be carried out very well on an industrial scale favorably using a suspension of conidiospores.
It is this property however which is lacking in the prior art strains of Claviceps which form alkaloids of the ergotoxine group in saprophytic cultures. This shortcoming appears clearly from Hungarian Pat. No. 150,631 and from AMICI et al. (Appl. Microbiology 18 (1969), pp. 464-468). The conditions for the formation of seed cultures on an industrial scale are not good in these cases because of the necessity for reproduction of the inoculum on solid substrata and for homogenization of the thus obtained mycelium.
The publications cited also show that up to the end of the fermentation process with these ergotoxine forming microorganism only a small portion of the ergotoxine which is formed in the cells will permeate the cell walls and enter the surrounding aqueous culture medium.
In order to carry out a complete production of the formed alkaloids the separate processing both of the culture filtrate and the mycelium is necessary. The high pigment and dead weight portion formed usually in strains which are adapted for biosynthesis of ergot alkaloids in submersed cultures requires highly complicated operations and results in unsatisfactory yields.
Thus, according to the British Pat. No. 1,158,380, the obtained culture filtrate is extracted with chloroform at pH 9, the obtained extract is then extracted with aqueous tartaric acid and the resulting aqueous extract is again extracted with chloroform at pH 9 and the latter extract is finally united with the mycelium extract which has been obtained and purified in a similar complicated manner, and is then concentrated by evaporation whereupon the crude alkaloid bases are finally precipitated with hexane. From the precipitate ergotamine sulfate is removed with glacial acetic acid, methanol and sulfuric acid, the residue is concentrated by evaporation, an aqueous solution thereof is extracted at pH 9 again with chloroform, the concentrated chloroform extract is subjected to chromatography through 100 times the amount of silica gel and the thus purified ergocryptine is finally isolated as base from benzene. By means of a further chromatographic fraction additional ergotamine can be obtained after concentration by evaporation and solution of the residue in aqueous acetone.
The directions of British Pat. No. 1,158,380 and U.S. Pat. No. 3,485,722 for isolating ergot peptide alkaloids from cultures of a new strain of Claviceps purpurea likewise had no success. Disturbing in this case was particularly the strong tendency of the culture filtrate and of the mycelium extract to form emulsions during chloroform extraction. Furthermore, it was not possible to convert the ergotoxin into a crystalline base and to obtain the yields of noncrystalline pure or crude alkaloid concentrates mentioned in the above patent.
The Pat. No. 41,967 of the German Democratic Republic describes a process wherein an extract which is obtained in known manner from a culture medium which contains a low alkaloid concentration is subjected to chromatography with increasingly polar eluants whereupon the individual alkaloids are crystallized in conventional manner. The extract in this case contained ergotoxin and ergometrine together. However, it is well known that the elution with methanol-containing solvents results in unsatisfactorily high pigment fractions in the ergometrine.
According to the German Pat. No. 883,153 and the Canadian Pat. No. 470,573 the pressure hydrogenation of ergotoxin is carried out with highly active palladium or carrier supported palladium. This type of process apparently due to an unsatisfactory selectivity resulted in undesirable pigmentation of the hydrogenation product which again required additional purification operations.
It is therefore an object of the invention to provide for a process for the industrial production of ergoline derivatives, in particular alkaloids and 9,10-dihydroalkaloids of the ergotoxin group and also of ergometrine which avoids these shortcomings.